The focus of this proposal is to examine molecular mechanisms that regulate protein synthesis in neurons in response to group 1 metabotropic receptor (mGluR) activation. mGluRI and 5 transduce excitatory synaptic stimuli to active phospholipase C and release Ca2+ from intracellular stores (1), and play an important role in regulating trafficking of AMPA and NMDA type ionotropic glutamate receptors (2, 3). mGluR 1 and 5 are also key regulators of protein synthesis at the excitatory synapse (4). Recently, it has been shown that mGluRS is essential for cocaine addiction (5) and other forms of protein synthesisdependent plasticity (6-10). Ongoing studies conducted during the previous grant period identified two distinct regulatory mechanisms that transduce mGluR activity to increase protein synthesis. The most rapid response is mediated by a molecular cascade that involves dynamic interactions between mGluR and eukaryotic elongation factor 2 kinase (eEF2K). Our interest in eEF2K began with the discovery that it binds both mGluR and Homer. Agonist stimulation of mGluR results in activation of eEF2K in a process that appears to involve calmodulin and requires intracellular stores of Ca2+. eEK2K phosphorylates elongation factor 2 (eEF2), which binds the polyribosome-mRNA complex and controls the elongation phase of protein translation (11). Phosphorylated eEF2 (p-eEF2) acts as a dominant negative and prevents translation of many mRNAs, although it is reported that p-eEF2 can selectively increase translation of certain mRNAs (12, 13). Time course studies indicate that activation of mGluR results in an immediate increase in p-eEF2 (should reduce elongation) that persists for ~5 min followed by a progressive decrease of p-eEF2 that persists >60 min. Our studies indicate that this pathway is essential for the rapid translation of the immediate early gene Arc. Neurons in which eEF2K is genetically deleted (ko) show an absence of the initial upregulation of Arc at 5 min after mGluR activation. Importantly, eEF2K ko mice phenocopy changes in plasticity seen in Arc ko mice. Arc is representative of a class of mRNAs (includes CamKII), that possess an internal ribosomal entry sequence (IRES) (14). Viral IRESs form a secondary structure that imitates the initiation complex required for ribosomal binding and therefore bypass regulation of initiation. We hypothesize that the IRES affords regulation by eEF2 in mGluR-dependent Arc translation, and that understanding of this process will reveal basic insights into synapse-specific protein synthesis. Arc is highly relevant for studies of addiction since it is strongly induced in accumbens/striatum by cocaine, methamphetamine, and dopamine receptor activation (15-20).